Ž .Bioelectrochemistry and Bioenergetics 48 1999 431–434

Multiple-pulse-mediated electrofusion of intact erythrocyte onto humanterm placental amnion

Subrata Biswas ), Sujoy K. GuhaCentre for Biomedical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India

Received 24 November 1998; received in revised form 26 January 1999; accepted 12 February 1999

Abstract

The creation of surface modified human term placental amnion by electrofusing human cells onto its surface has been thought of. Amultiple-pulse electrofusion protocol with 10 square pulses of 10-ms pulse length, and electric field of 0.2 kV cmy1, can makeerythrocyte–amnion tissue electrofusion possible. The protocol devised merge the cell-tissue-adherence steps with fusogenic pulse. Thefinding opens up a new avenue of cell electrofusion onto human tissue with minimal procedural complexities. q 1999 Elsevier ScienceS.A. All rights reserved.

Keywords: Microvilli; Placental amnion; Cell-tissue electrofusion; Graft tissue

1. Introduction

Incorporation of functional cell membrane to a humangraft tissue would broaden the scope of investigations onthe fused cell membrane function. Thus, experimentationon human cells, especially with its membrane, can becomeeasier when it will be available on graft membrane surface.A novel animal model developed by this technique has

w xalready been demonstrated 1–4 . In these cell-tissue elec-Ž .trofusion CTE experiments human and nonhuman cul-

tured cells were electrofused directly onto rabbit cornea.There is also a need to find similar fusion receptacles onsome human tissues so that in the future this electromanip-ulation technique may be useful in developing human CTEor tissue–tissue electrofusion, which may find its applica-tions in processes like wound healing, surface modificationof human tissues, etc. Human amnion or intact placental

w xmembrane, a heterograft membrane 5 , may serve as agood receptacle of human cells on its surface. This tissuehas been selected as a model human tissue for CTEbecause placental amnion is formed from the ectoderm of

w xthe fetus; thus it is like an extension of skin 5 . Moreover,unlike skin tissue, placental tissue is nonimmunogenic,hence excludes the possibility of artifact fusion. Thus

Ž .isolated cells fusion partner are normally not expected tofuse or adhere onto its surface. In addition, its isolated

) Corresponding author

placental amnion cells in culture respond to electromanipu-lation, and the required electrical parameters are knownw x6 . Human erythrocyte is selected as the fusion partnersince much is known about its membrane and many proto-

w xcols have been developed for use with it 7–9 .Human amnion cells in culture require field pulse treat-

y1 Ž .ment of 0.2 to 0.3 kV cm and duration t 400 ms forw xmembrane breakdown 6 , whereas human erythrocytesŽ . y1need a pulse electric field E of 1.5 to 6.0 kV cm and t

w xof 1 ms 7,8 . Clearly the two cells require differentelectro-pulsing conditions. It is speculated that both thecells simultaneously can be made fusogenic by usingelectric-field pulses of short duration in succession. Multi-ple pulsing may also lead to dielectrophoresis-mediatedcell-tissue contact formation.

This investigation is aimed to develop a protocol ofmultiple-pulse CTE, for fusion of human erythrocytes ontoamnion of human term placenta, without any attempt tocontact the cells to the tissue before or after pulsing.

2. Materials and methods

2.1. Cell and tissue preparation

Freshly obtained heparinized human blood was washedŽ .three times by centrifugation 2500 rpm for 10 min in

Ž .isotonic phosphate buffer NaPi at room temperature.

0302-4598r99r$ - see front matter q 1999 Elsevier Science S.A. All rights reserved.Ž .PII: S0302-4598 99 00042-2

( )S. Biswas, S.K. GuharBioelectrochemistry and Bioenergetics 48 1999 431–434432

Buffy coat was removed by aspiration and the pellet wasresuspended to a concentration of 10=108 cells mly1 in

ŽRinger solution 20 mM KH PO , 46.7 mM Na HPO , 702 4 2 4.mM NaCl, pH 7.4 .

Human full-term placental membrane was collected im-mediately after delivery and washed thoroughly with Ringersolution to remove blood. The amnion cell layer was splitoff. A small piece of the tissue was stretched and mountedin between a double lucite chamber.

2.2. Cell-tissue electrofusion

The CTE chamber was specially designed to preventtissue damage. Each side of the chamber was lined with asoft, natural rubber gasket and layered with silicone greaseto prevent leaks and tissue damage due to the compression.On the maternal side of the tissue 0.3 ml of Ringer, and onthe fetal side 0.3 ml of the RBC suspension were addedand allowed to settle on the tissue for ;3 min. Twostainless-steel plate electrodes of dimension, 1.5 cm=2cm, were attached to either side of the chambers andconnected to the output of a DC power supply, and aswitching circuit. Pulsing across the sample for particularduration of time was determined by electronic switch. Thetransistor switching circuit was driven by selected voltagerectangular pulse from a waveform generator. Ten 0.2-kV

y1 Žcm rectangular pulses as rectangular pulses can bedelivered more conveniently as pulse train, compared to

.exponential-decay pulse with 1-Hz frequency and dura-

Ž .tion t 10 ms were administered. Electromanipulatedsamples were then processed for electron microscopy.

2.3. Scanning electron microscopy

Tissues were washed thoroughly in Ringer and fixedovernight in 2.5% glutaraldehyde. After several washes ingraded acetone and two changes in absolute alcohol thesamples were critical point dried at 0.70 bar pressure in

w xCO 10 . Dried samples were coated with gold in a2

Polaron scanning electron microscopic coating system, andŽ .examined in a Cambridge stereo-scan S-360 scanning

electron microscope.

3. Results and discussion

Ž .Fig. 1 shows the scanning electron microscopy SEMimage of human term placental amnion surface. The cellsurface is characterized by numerous microvilli. On thistissue surface human RBC were electrofused using multi-ple-pulse protocol. Fig. 2 shows an SEM image of twoblood cells fused onto placental amnion surface. The mem-branes of RBC and microvilli were clearly fused overmany areas along the circumference of the RBC. One ofthe fused red cells retained its normal disc shape even afterfusion with microvilli. Other cells showed deviation fromthe normal shape. Differences in morphology of the twofused blood cells might be an indication of the initiation of

Fig. 1. SEM image of human term placental amnion at the fetal side. Surface of the tissue has prominent microvilli.

( )S. Biswas, S.K. GuharBioelectrochemistry and Bioenergetics 48 1999 431–434 433

Fig. 2. Human erythrocyte electrofused on top of the amnion tissue surface. Erythrocyte membrane appears to have fused with microvilli membrane.

fusion at different times. Thus the rod-shaped microvillihave completely fused with the red cell membrane provingthe effectiveness of the multiple pulse hetero-electrofusion.Fig. 2 also shows unfused microvilli, which are separated

Ž .from each other just like control samples Fig. 1 . Cobble-stone effect similar to that observed in microvilli of

w xmacrophage isoelectrofusion 11 is not evident.ŽThe cell surface morphology of placental amnion Fig.

.1 makes it difficult to carry out theoretical calculationsand to model the cell. Moreover, heterofusion of two cellsstudied here differ in morphology which makes the calcu-lation more complicated. Experimental investigation re-veals that 10 rectangular pulses of 0.2 kV cmy1 fieldstrength and duration 10 ms can induce fusion of humanerythrocytes with amnion tissue. The multiple pulsing canresult in CTE without adherence step. The process may behelpful in the development of human tissue models, butwhether cell-tissue membrane adherence leading to CTEwas a pre-pulse or post-pulse event is not clear.

A system of microvilli was observed in the intercellularw xspace of amnion tissue 12 , similar to spontaneous clump-

w xing of chicken embryonic retina cells 13 , adhesion ofw xmammalian blood platelets 14 , and limpet haematocytes

w x15,16 , indicating strong intercellular adhesion. Thus, thereis a possibility that the microvilli on the surface of amnioncells may also initiate pre-pulse membrane-to-membranecontact with neighboring erythrocyte membrane. Howeversuch adhesion is initiated with active participation of mem-brane extension. Erythrocyte membrane microextension

y2 w xrequires a minimum of 0.2 to 0.3 mJ m of energy 17 .The intercellular distance between the two approaching

˚membranes without any external aidss is more than 250 A

w x18 . At this intercellular distance, van der Waals attractionŽ .potential which might involve in membrane perturbation

is less than 10y4 mJ my2 , which is very low compared tothe energy required for erythrocyte microextension forma-tion. Thus erythrocytes are very unlikely to produce cyto-plasmic extension. This confirms that there is no possibil-ity of cytoplasmic extension mediated pre-pulse cell-tissuecontact formation.

Amnion cell microvilli might be important in penetra-tion of the electrostatic repulsion as low diameter projec-

w xtion on P815Y Mastocytoma cells 19 do. If the meanlength of the amnion microvilli is assumed to be the same

w xas that of P815Y Mastocytoma, that is, 0.4 mm 16 , andwidth 0.25 mm, then a reasonable estimate of their surfacearea is about 1.0=10y13 m2. On the other hand theadhesion force required for mechanically bringing two

y13 y11 w xcells in contact ranges from 10 to 10 N 20 . Thepressure exerted on the microvilli by the force driving twocells towards each other is about:

10y13 Ny10y11 N2 y2s1y10 N my13 210 m

The estimated force is lower than the intercellular vander Waals attraction and electrostatic repulsion at intercel-

˚ w xlular distance, more than 250 A 20 . Moreover, erythro-cyte surfaces do not have such microvilli. Thus the ex-pected pressure is even lower than the estimated pressure.Hence, the possibility of microvilli penetrating electrostaticrepulsion is ruled out.

Pulse trigger movement for free cells might be the main˚impetus to the cell-tissue approach below 250 A, and lead

( )S. Biswas, S.K. GuharBioelectrochemistry and Bioenergetics 48 1999 431–434434

Ž .to membrane contact. The pulse driven distance D cov-Ž . w xered by the cell can be determined from Eq. 1 21 :

2 < < 2R KNt = ECellDs 1Ž .

3h

where R is the radius of the cell, K is dielectriccell

constant, N is number of pulses, t is pulse duration, Eelectric field, and h is the viscosity of the medium. If< < 2 2 y1E sE relectrode gap, Es0.2 kV cm , ts10 ms,hf0.894=10y3 Pa s and Ns10, the displacement of

˚cells during the pulse is found to be ;0.04 A. Thedistance covered during the pulse is less than that of the

˚Ž .expected pre-pulse cell-tissue gap )250 A , indicatingcell-tissue contact formation and further fusion reactionswere initiated after the pulse was over. Most likely, postpulse membrane-to-membrane interactions, like van derWaals and modified electrostatic interactions may play animportant role in adequate juxtapositioning and two mem-branes merger.

Acknowledgements

The authors are grateful to Prof. S. Anand, CBME, IITDelhi, for providing opportunity to work in her laboratory.S. Biswas was a recipient of a doctoral fellowship of theministry of HRD, India.

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